Mechanistically diverse superfamilies provide the opportunity to understand the structural bases for divergent evolution of enzyme function. In the enolase superfamily, the reactions are initiated by abstraction of the a-proton of a carboxylate anion substrate to yield a Mg^^-stabilized enolate intermediate; the intermediate is directed to product by an appropriately located active site acid. In the RuBisCO superfamily, the reactions are initiated by abstraction of the a-proton of a ketose 1-phosphate substrate to yield a Mg^'^-stabilized enolate intermediate; the potential fate(s) of the intermediate are pooriy understood but may involve tautomerization, dehydration, oxidation, and/or carboxylation. This project describes structure/function aspects of our integrated sequence-structure-computation strategy for predicting the substrates specificities and, therefore, assigning functions of uncharacterized proteins in both superfamilies. The focus is on proteins that are encoded by operons: the enzymes that catalyze successive steps in a metabolic pathway should share conserved elements of substrate specificity, thereby facilitating identification of the functions of all of the enzymes in the pathway and, therefore, new metabolism. The project is organized in three Specific Aims: Specific Aim 1 focuses on divergent members of the muconate lactoniziing enzyme subgroup of the enolase superfamily (Lys acid/base catalysts at the ends of the second and sixth (J-strands of the barrel domain), including 1) dipeptide epimerases that are encoded by operons that also encode homologues of dipeptidases, and 2) two novel subgroups whose members are expected to catalyze new reactions. Specific Aim 2 focuses on divergent members of the mandelate racemase subgroup of the enolase superfamily (an acid/base His-Asp dyad at the ends of the seventh and sixth p-strands of the TIM-barrel domain) that are encoded by operons, with these also encoding aldolases, dehydrogenases, mutarotases, and/or kinases. Specific Aim 3 focuses on RuBisCO-like proteins (RLPs) that are encoded by operons that also encode homologues of isomerases, aldolases, transketolases, and other aldose/ketose 5-phosphate utilizing enzymes. RELEVANCE (See instructions); The assignment of functions to the complete set of proteins encoded by genomes is a major problem. However, when this problem is solved, their roles in molecular, cellular, and organismal functions will be known and novel targets for specific small molecule intervention can be identified, thereby providing new approaches for therapeutic design. This Program Project is focused on developing and implementing an integrated sequence-structure-computation strategy for predicting the substrate specificities of uncharacterized proteins discovered in genome projects, thereby facilitating their functional assignment.